Antibody-serum protein hybrids

ABSTRACT

Hybrid proteins are described which comprise one or more antigen-binding antibody fragments covalently linked to one or more serum carrier proteins. The hybrid proteins can bind antigens, have a long half-life in vivo and can be used in medicine for therapy and diagnosis.

This invention relates to modified antibody fragments, to processes fortheir preparation and to their use in medicine.

Since antibodies show great specificity in their binding to othermolecules, they are of benefit as therapeutic or diagnostic agents, oras reagents (for example, as affinity purification reagents or ascatalytic enzymes). The advent of hybridoma technology and thegeneration of transgenic animals has allowed the production of largevolumes of monoclonal antibodies, sufficient for therapeutic use. Mostsuch antibodies have been generated for treatment of acute diseases,such as particular types of cancer. For the treatment of chronic and/ormore common diseases, still larger amounts of antibody are likely to berequired. An increased usage also brings an increased need for a cheapertherapeutic agent.

Monoclonal antibodies can be produced in cultured mammalian or insectcells, or in transgenic animals, but the ability of these technologiesto supply a large market at a reasonable cost, is unproven so far.Again, fungi have been shown to be able to produce heterologous proteins[e.g. Sleep, D., et al (1991) Bio/Technology, 9, 183-187], butexpression of whole immunoglobulin G (IgG1) in a fungus has beenreported to occur only at low level (in Saccharomyces cerevisiae), or inshakeflask culture (in Pichia pastoris), so antibody production fromfungi on an industrial scale is unproven. [Horwitz, A. H., et al (1988)Proc. Natl. Acad. Sci. USA, 85, 8678-8682; Ogunjimi, A. A. et al (1999)Biotechnology Lett. 21, 561-567]. Furthermore, glycosylation of theantibody would be expected to be unlike that from mammalian systems, andin mammals this may generate problems of immunogenicity and abnormalfunction (complement activation, binding to Fc receptors, transcytosisand prolongation of half-life through interaction with FcRn receptor)[Nose, M. and Wigzell, H. (1983) Proc. Natl. Acad. Sci. USA, 80,6632-6636; Tao, M.-H. and Morrison, S. L. (1989) J. Immunol. 143,2595-2601; Wawrzynczak, E. J. et al (1992) Molec. Immunol. 29, 213-220;Kim, J.-K., et al (1994) Eur. J. Immunol. 24, 2429-2434].

Bacterial systems are not known to be capable of producing a whole,functioning antibody in a yield sufficient to give an economic processfor large-scale manufacture, but they are a source of low-costimmunoglobulin fragments, such as Fab′ [Better, M., et al. (1988)Science, 240, 1041-1043]. Antibody fragments, however, may lack variousof the functions of whole antibody. For example, Fab′, F(ab′)₂ or scFvlack the Fc domain that imparts a long lifetime in vivo: [Medesan, C. etal (1997) J. Immunol. 158, 2211-2217]. The half-life in circulation inmammals of Fab′ or F(ab′)₂ has been reported as being about 1% that ofwhole IgG [Waldmann, T. A. and Strober, W. (1969) Progr. Allergy, 13,1-110.], and the β-phase half-life (the time taken for half of themolecules in circulation to be eliminated) of Fab′ has been reported asbeing about 5% that of whole IgG [Chapman, A. P., et al (1999) NatureBiotechnology, 17, 780-783]. Thus, while they may be cheap to produce,these fragments are eliminated rapidly from circulation and can be oflimited therapeutic use. This has led to attempts to prolong half-lifeof antibody fragments, for instance by modification of Fab′ or F(ab′)₂in vitro by addition of one or more molecules of polyethylene glycol toeach fragment molecule. International Patent Specification No.WO98/925971

In the present invention we have addressed the identifiable need for ameans to economically produce an IgG fragment that can bind antigen andthat has a long half-life in vivo. We have achieved this by employing acarrier protein to prolong the immunoglobulin in circulation.

Thus according to one aspect of the invention we provide amulti-component hybrid protein comprising one or more antigen-bindingantibody fragments covalently linked to one or more serum carrierproteins or fragments thereof.

A variety of proteins exist in plasma, circulating in the body withhalf-lives measured in days, for example, 5 days for thyroxine-bindingprotein or 2 days for transthyretin [Bartalena, L. and Robbins, J.(1993) Clinics in Lab. Med. 13, 583-598], or 65h in the second phase ofturnover of iodinated α1-acid glycoprotein (Bree, F. et al (1986) Clin.Pharmacokin. 11, 336-342]. Again, data from Gitlin et al. [Gitlin, D.,et al (1964) J. Clin. Invest. 10, 1938-1951] suggest that in pregnantwomen the half-life of α1-acid glycoprotein is 3.8 days, 12 days fortransferrin and 2.5 days for fibrinogen. Serum albumin is an abundantprotein in both vascular and extravascular compartments [Peters, Jr., T.(1985) Adv. Prot. Chem. 37, 161-245]. The half-life of albumin in man,about 19 days [Peters, 1985 ibid], is similar to that of IgG1 (about 21days [Waldeman+Strober ibid], though it is less in other species—about 2days in rats, for example [Peters, 1985 ibid]. Albumin does not possessthe noted ability of antibodies to specifically bind ligands,particularly those of high molecular weight.

In the present invention we have produced a series of hybrid proteinsthat advantageously have the antigen-binding capabilities of an antibodyfragment and the longevity of serum albumin in vivo. Additionally thehybrids may be especially suitable for use in expectant or nursingmothers since as with various other serum proteins, albumin istransported poorly across the placenta: labelled albumin injected into amother appears with 5% or less specific activity in the foetus after 25days [Gitlin, D. Kumate, J. Urrusti, J. and Morales, C. (1964) J. Clin.Invest. 10, 1938-1951]. This contrasts with IgG, which is transportedefficiently across the placenta to the foetus. Similarly, albumin is nottransported across the gut wall of neonates, whereas IgG is, byinteraction with the FcRn receptor. Thus advantageously a foetus orneonate would be exposed to minimal amounts of the hybrids according tothe invention from maternal circulation or milk.

With regard to commercial production, recombinant albumin has beenreported as being produced at several gm per litre of culture of yeast(Pichia pastoris or Kluyveromyces lactis [Barr, K. A., et al (1992)Pharm. Eng. 12, 48-51; Fleer, R., et al (1991) Bio/Technology 9,968-975; Cregg, J. M., et al (1993) Bio/Technology 11, 905-910]). Thislevel of expression makes industrial production of pharmaceutical gradeprotein economically feasible, as remarked by Fleer et al [Fleer, R., etal (1991) Bio/Technology 9, 968-975]. Similarly, transgenic mice havebeen found to express as much as 10 gm per litre of albumin in theirmilk [Hurwitz, D. R, et al (1994) Transgenic Res. 3,365-375].

Depending on the intended specific use and/or half-life required, thehybrid protein according to the invention may be in a number ofdifferent forms. For example, one protein according to the invention maycomprise an antibody fragment covalently linked to two, three or moreserum carrier proteins or fragments thereof each of which may be thesame or different. In another example, a protein according to theinvention may comprise two, three or more antibody fragments, which maybe the same or different, each covalently linked to the same serumprotein or a fragment thereof. In general, however, a preferred hybridprotein according to the invention comprises one, two or threeantigen-binding antibody fragments covalently linked to one serumcarrier protein or a fragment thereof.

Each antigen-binding antibody fragment component in the proteinsaccording to the invention will in general comprise an antibody variableregion domain containing one or more antigen binding sites.

The variable region domain may be of any size or amino acid compositionand will generally comprise at least one hypervariable amino acidsequence responsible for antigen binding embedded in a frameworksequence. In general terms the variable (V) region domain may be anysuitable arrangement of immunoglobulin heavy (V_(H)) and/or light(V_(L)) chain variable domains. Thus for example the V region domain maybe monomeric and be a V_(H) or V_(L) domain where these are capable ofindependently binding antigen with acceptable affinity. Alternativelythe V region domain may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L),or V_(L)-V_(L), dimers in which the V_(H) and V_(L) chains arenon-covalently associated. Where desired, however, the chains may becovalently coupled either directly, for example via a disulphide bondbetween the two variable domains, or through a linker, for example apeptide linker, to form a single chain domain, e.g. scFv.

The variable region domain may be any naturally-occurring variabledomain or an engineered version thereof. By engineered version is meanta variable region domain that has been created using recombinant DNAengineering techniques. Such engineered versions include those createdfor example from natural antibody variable regions by insertions,deletions or changes in or to the amino acid sequences of the naturalantibodies. Particular examples of this type include those engineeredvariable region domains containing at least one CDR and optionally oneor more framework amino acids from one antibody and the remainder of thevariable region domain from a second antibody.

The variable region domain will in general be capable of selectivelybinding to an antigen. The antigen may be any cell-associated antigen,for example a cell surface antigen such as a T-cell, endothelial cell ortumour cell marker, or it may be an extracellular matrix antigen, anintracellular antigen or a soluble antigen. Particular examples of cellsurface antigens include adhesion molecules, for example integrins suchas β1 integrins, e.g. VLA-4, E-selectin, P-selectin or L-selectin, CD2,CD3, CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25,CD33, CD38, CD40, CD45, CDW52, CD69, carcinoembryonic antigen (CEA),human milk fat globulin (HMFG1 and 2), MHC Class I and MHC Class IIantigens, and VEGF, and where appropriate, receptors thereof. Solubleantigens include interleukins such as IL-1, IL-2, IL-3, IL4, IL-5, IL-6,IL-8 or IL-12, viral antigens, for example respiratory syncytial virusor cytomegalovirus antigens, immunoglobulins, such as IgE, interferonssuch as interferon-α, interferon-β or interferon-γ, tumour necrosisfactor-α, tumour necrosis factor-β, colony stimulating factors such asG-CSF or GM-CSF, and platelet derived growth factors such as PDGF-α, andPDGF-β and where appropriate receptors thereof.

In practice it is generally preferable that the variable region domainis covalently attached at a C-terminal amino acid to at least one otherantibody domain or a fragment thereof. Thus, for example where a V_(H)domain is present in the variable region domain this may be linked to animmunoglobulin C_(H)1 domain or a fragment thereof. Similarly a V_(L)domain may be linked to a C_(K) domain or a fragment thereof. In thisway for example the fragment according to the invention may be a Fabfragment wherein the antigen binding domain contains associated V_(H)and V_(L) domains covalently linked at their C-termini to a C_(H)1 andC_(K) domain respectively. The CH1 domain may be extended with furtheramino acids, for example to provide a hinge region domain as found in aFab′ fragment, or to provide further domains, such as antibody CH2 andCH3 domains.

Each serum carrier protein component in the hybrid proteins according tothe invention may be a naturally occurring serum carrier protein or afragment thereof. Particular examples include thyroxine-binding protein,transthyretin, α1-acid glycoprotein, transferrin, fibrinogen and,especially, albumin, together with fragments thereof. The carrierproteins will in particular be of human origin. Where desired each mayhave one or more additional or different amino acids to the naturallyoccurring sequence providing always that the resulting sequence isfunctionally equivalent with respect to half-life. Fragments include anysmaller part of the parent protein that retains the carrier function ofthe mature sequence.

The antibody and carrier protein components in the hybrid proteinsaccording to the invention may be directly or indirectly covalentlylinked. Indirect covalent linkage is intended to mean that an amino-acidin an antibody fragment is attached to an amino-acid in a carrierprotein through an intervening chemical sequence, for example a bridginggroup. Particular bridging groups include for example aliphatic,including peptide, chains as more particularly described hereinafter.Direct covalent linkage is intended to mean that an amino acid in anantibody fragment is immediately attached to an amino acid in a carrierprotein without an intervening bridging group. Particular examplesinclude disulphide (—S—S—] and amide [—CONH—] linkages, for example whena cysteine residue in one component is linked to a cysteine residue inanother through the thiol group in each, and when the C-terminal acidfunction of one component is linked to the N-terminal amine of theother.

Particular bridging groups useful to indirectly link an antibody to acarrier protein include optionally substituted aliphatic,cycloaliphatic, heteroaliphatic, heterocycloaliphatic, aromatic andheteroaromatic groups. Particular groups include optionally substitutedstraight or branched C₁₋₂₀alkylene, C₂₋₂₀alkenylene or C₂₋₂₀alkynylenechains optionally interrupted and/or terminally substituted by one ormore —O— or —S— atoms, or —N(R¹)— [where R¹ is a hydrogen atom or aC₁₋₆alkyl group], —N(R¹)CO—, —CON(R¹)—, —N(R¹)SO₂—, —SO₂N(R¹)—, —C(O)—,—C(O)O—, —OC(O)—, —S(O)—, —S(O)₂—, aromatic, e.g. phenyl,hereroaromatic, e.g. pyridyl, or cycloalkyl, e.g. cyclohexyl groups.Such chains include for example optionally substituted straight orbranched C₁₋₁₀alkylene chains such as optionally substituted butylene,pentylene, hexylene or heptylene chains, single amino acid residues andpeptide chains, for example containing two to twenty amino acids, whichmay be the same or different, e.g. polyglycine chains such as(Gly)_(n)where n is an integer from two to ten. Optional substituentswhich may be present on any of the above chains include one or morehalogen atoms, e.g. fluorine, chlorine, bromine or iodine atoms, orC₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆aloalkyl, C₁₋₆haloalkoxy, —OH or —N(R¹)(R²)[where R² is as defined for R¹] groups.

Where a bridging group indirectly links an antibody to a carrier proteinthe linkage may be to the side chain of any suitable amino acid, forexample a lysine, arginine, serine, aspartic acid, glutamic acid orcysteine residue, located in the antibody or carrier protein. At eachpoint of attachment the residue of a reactive group may be present. Forexample, where the bridging group is linked to a cysteine residue in theantibody or carried protein, the residue of a thiol-selective reactivegroup such as a maleimide group or the like may be incorporated as partof the attachment.

Where desired, the hybrid protein according to the invention may haveone or more effector or reporter molecules attached to it and theinvention extends to such modified proteins. The effector or reportermolecules may be attached to the antibody fragment and/or the carrierprotein through any available amino acid side-chain or terminal aminoacid functional group located in either component, for example any freeamino, imino, hydroxyl or carboxyl group. The linkage may be direct orindirect, through spacing or bridging groups, as just described above,for linking the antibody and carrier protein components. Alternativelythe reporter/effector may be attached to the linking moiety

Effector molecules include, for example, antineoplastic agents, toxins(such as enzymatically active toxins of bacterial or plant origin andfragments thereof e.g. ricin and fragments thereof) biologically activeproteins, for example enzymes, nucleic acids and fragments thereof, e.g.DNA, RNA and fragments thereof, natural or synthetic polymers such aspolysaccharides or polyalkylene polymers such as poly(ethylene glycol),radionuclides, particularly radioiodide, and chelated metals. Suitablereporter groups include chelated metals, fluorescent compounds orcompounds which may be detected by NMR or ESR spectroscopy.

Particular antineoplastic agents include cytotoxic and cytostaticagents, for example alkylating agents, such as nitrogen mustards (e.g.chlorambucil, melphalan, mechlorethamine, cyclophosphamide, or uracilmustard) and derivatives thereof, triethylenephosphoramide,triethylenethiophosphoramide, busulphan, or cisplatin; antimetabolites,such as methotrexate, fluorouracil, floxuridine, cytarabine,mercaptopurine, thioguanine, fluoroacetic acid or fluorocitric acid,antibiotics, such as bleomycins (e.g. bleomycin sulphate), doxorubicin,daunorubicin, mitomycins (e.g. mitomycin C), actinomycins (e.g.dactinomycin) plicamycin, calichaemicin and derivatives thereof, oresperamicin and derivatives thereof; mitotic inhibitors, such asetoposide, vincristine or vinblastine and derivatives thereof;alkaloids, such as ellipticine; polyols such as taxicin-I or taxicin-II;hormones, such as androgens (e.g. dromostanolone or testolactone),progestins (e.g. megestrol acetate or medroxyprogesterone acetate),estrogens (e.g. dimethylstilbestrol diphosphate, polyestradiol phosphateor estramustine phosphate) or antiestrogens (e.g. tamoxifen);anthraquinones, such as mitoxantrone, ureas, such as hydroxyurea;hydrazines, such as procarbazine; or imidazoles, such as dacarbazine.

Particularly useful effector groups are calichaemicin and derivativesthereof (see for example South African Patent Specifications Nos.85/8794, 88/8127 and 9012839).

Chelated metals include chelates of di-or tripositive metals having acoordination number from 2 to 8 inclusive. Particular examples of suchmetals include technetium (Tc), rhenium (Re), cobalt (Co), copper (Cu),gold (Au), silver (Ag), lead (Pb), bismuth (Bi), indium (In), gallium(Ga), yttrium (Y), terbium (Tb), gadolinium (Gd), and scandium (Sc). Ingeneral the metal is preferably a radionuclide. Particular radionuclidesinclude ^(99m)Tc, ¹⁸⁶Re, ¹⁸⁸Re, ⁵⁸Co, ⁶⁰Co, ⁶⁷Cu, ¹⁹⁵Au, ¹⁹⁹Au, ¹¹⁰Ag,²⁰³Pb, ²⁰⁶Bi, ²⁰⁷Bi, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ⁸⁸Y, ⁹⁰Y, ¹⁶⁰Tb, ¹⁵³Gd and ⁴⁷Sc.

The chelated metal may be for example one of the above types of metalchelated with any suitable polydentate chelating agent, for exampleacyclic or cyclic polyamines, polyethers, (e.g. crown ethers andderivatives thereof); polyamides; porphyrins; and carbocyclicderivatives.

In general, the type of chelating agent will depend on the metal in use.One particularly useful group of chelating agents in conjugatesaccording to the invention, however, are acyclic and cyclic polyamines,especially polyaminocarboxylic acids, for examplediethylenetriaminepentaacetic acid and derivatives thereof, andmacrocyclic amines, e.g. cyclic tri-aza and tetra-aza derivatives (forexample as described in International Patent Specification No. WO92/22583); and polyamides, especially desferrioxamine and derivativesthereof.

Particularly useful hybrid proteins according to the invention includeone or more antigen-binding antibody fragments covalently linked to oneor more albumin molecules or fragments thereof.

Particular hybrids of this type include those wherein oneantigen-binding antibody fragment is covalently linked to an albuminmolecule or a fragment thereof. In these hybrids, and in general in theproteins according to the invention, each antigen-binding antibodyfragment is preferably a monovalent Fab′ fragment, optionally containingone or more additional amino acids attached to the C-terminus of the CH1domain and is especially a Fab′ fragment. Particularly useful Fab′fragments include those wherein the hinge domain contains a singlecysteine residue. Fragments of albumin include one or more of domains I,II and/or III or subdomains thereof [see for example Peters, T. in “Allabout Albumin”, Academic press, London (1996)].

Especially useful antibody-albumin hybrids according to the inventioninclude those in which each protein component is directly linked throughthe C-terminal amino acid of the antibody to the N-terminal amino acidof the albumin. Where desired, one or more, e.g. up to around 100,additional amino acids may be inserted between the C— and N-termini toform a spacing group.

Another particularly useful class of antibody-albumin hybrids accordingto the invention is that wherein each protein component is indirectlylinked between the thiol groups of a cysteine residue present in theantibody and another in the albumin. The indirect linkage may beachieved by a bridging molecule as described above. Particularly usefulgroups include non-cleavable linker groups, especially optionallysubstituted straight or branched C₁₋₁₀ alkylene chains.

In this class of hybrids the antibody is preferably a Fab′ fragmentoptionally containing one or more additional amino acids attached to theC-terminal of the CH1 domain. Especially useful fragments include Fab′fragments. The cysteine residue to which the spacing or bridgingmolecule is attached is preferably located in the CH1 domain of the Fabor, especially, is located in any C-terminal extension of the CH1 domainof the Fab, for example in the hinge domain of a Fab′.

The albumin in this class of hybrids may be in particular mature humanserum albumin or a fragment thereof. In this instance, the bridgingmolecule may be attached to the cysteine residue at position 34 of thealbumin. Advantageously, to avoid undesirable homodimer formation (seethe Examples below] the bridging molecule may be from around 10 Å toaround 20 Å in length, for example around 16 Å. Suitable bridgingmolecules in this length range may be easily determined from publishedsources, for example manufacturers' catalogues [see below]. Particularlyuseful bridging molecules include optionally substituted hexylenechains. Where each end of the bridging molecule is attached to thecysteine residue this may be through a disulphide bond or, inparticular, a sulphur-carbon bond. Where the linkage is a sulphur-carbonbond, the residue of a thiol-selective reactive group, such as amaleimide, may be present as part of each end of the spacing or bridginggroup.

The hybrid proteins according to the invention may be useful in thedetection or treatment of a number of diseases or disorders. Suchdiseases or disorders may include those described under the generalheadings of infectious disease, e.g. viral infection; inflammatorydisease/autoimmunity e.g. rheumatoid arthritis, osteoarthritis,inflammatory bowel disease; cancer; allergic/atopic disease e.g. asthma,eczema; congenital disease, e.g. cystic fibrosis, sickle cell anaemia;dermatologic disease, e.g. psoriasis; neurologic disease, e.g. multiplesclerosis; transplants e.g. organ transplant rejection,graft-versus-host disease; and metabolic/idiopathic disease e.g.diabetes.

The hybrid protein according to the invention may be formulated for usein therapy and/or diagnosis and according to a further aspect of theinvention we provide a pharmaceutical composition comprising amulti-component hybrid protein comprising one or more antigen-bindingantibody fragments covalently linked to one or more serum carrierproteins or fragments thereof, together with one or morepharmaceutically acceptable excipients, diluents or carriers.

As explained above, the hybrid protein in this aspect of the inventionmay be optionally linked to one or more effector or reporter groups.

The pharmaceutical composition may take any suitable form foradministration, e.g. for oral, buccal, parenteral, nasal, topical orrectal administration, or a form suitable for administration nyinhalation or insufflation, and preferably is in a form suitable forparenteral administration e.g. by injection or infusion, for example bybolus injection or continuous infusion. Where the composition is forinjection or infusion, it may take the form of a suspension, solution oremulsion in an oily or aqueous vehicle and it may contain formulatoryagents such as suspending, preservative, stabilising, antioxidant and/ordispersing agents.

Alternatively, the composition may be in dry form, for reconstitutionbefore use with an appropriate sterile liquid.

If the composition is suitable for oral administration the formulationmay contain, in addition to the active ingredient, additives such as:starch e.g. potato, maize or wheat starch or cellulose or starchderivatives such as microcrystalline cellulose; silica; various sugarssuch as lactose; magnesium carbonate and/or calcium phosphate. It isdesirable that, if the formulation is for oral administration it will bewell tolerated by the patient's digestive system. To this end, it may bedesirable to include in the formulation mucus formers and resins. It mayalso be desirable to improve tolerance by formulating the antibody in acapsule which is insoluble in the gastric juices. It may also bepreferable to include the antibody or composition in a controlledrelease formulation.

If the composition is suitable for rectal administration the formulationmay contain a binding and/or lubricating agent; for example polymericglycols, gelatins, cocoa-butter or other vegetable waxes or fats.

Therapeutic and diagnostic uses of hybrid proteins according to theinvention typically comprise administering an effective amount of theprotein to a human subject The exact amount to be administered will varyaccording to the intended use of the protein and on the age, sex andcondition of the patient but may typically be varied from about 0.1 mgto 1000 mg for example from about 1 mg to 500 mg. The protein may beadministered as a single dose or in a continuous manner over a period oftime. Doses may be repeated as appropriate. Typical doses may be forexample between 0.1-50 mg/kg body weight per single therapeutic dose,particularly between 0.1-20 mg/kg body weight for a single therapeuticdose.

The hybrid proteins according to the invention may be prepared bystandard chemical, enzymatic and/or recombinant DNA procedures.

Thus for example the hybrid protein may be prepared by the use ofrecombinant DNA techniques involving the manipulation and re-expressionof DNA encoding antibody variable and/or constant regions and a desiredcarrier protein or a fragment thereof. Such DNA is known and/or isreadily available from DNA libraries including for examplephage-antibody libraries [see Chiswell, D J and McCafferty, J. Tibtech.10, 80-84 (1992)] or where desired can be synthesised. Standardmolecular biology and/or chemistry procedures may be used to sequenceand manipulate the DNA, for example, to introduce codons to createcysteine residues, to modify, add or delete other amino acids or domainsin the antibody and/or carrier protein as desired.

From here, one or more replicable expression vectors containing the DNAmay be prepared and used to transform an appropriate cell line, e.g. anon-producing myeloma cell line, such as a mouse NSO line or abacterial, e.g. E. coli line, or, especially, a fungal line, such as ayeast line, e.g. members of the genera Pichia, Saccharomyces, orKluyveromyces, in which production of the antibody will occur. In orderto obtain efficient transcription and translation, the DNA sequence ineach vector should include appropriate regulatory sequences,particularly a promoter and leader sequence operably linked to thevariable domain sequence. Particular methods for producing recombinantproteins in this way are generally well known and routinely used. Forexample, basic molecular biology procedures are described by Maniatis etal [Molecular Cloning, Cold Spring Harbor Laboratory, New York, 1989];DNA sequencing can be performed as described in Sanger et al [PNAS 74,5463, (1977)] and the Amersham International plc sequencing handbook;and site directed mutagenesis can be carried out according to the methodof Kramer et al [Nucl. Acids Res. 12, 9441, (1984)] and the AnglianBiotechnology Ltd handbook. Additionally, there are numerouspublications, including patent specifications, detailing techniquessuitable for the preparation of proteins by manipulation of DNA,creation of expression vectors and transformation of appropriate cellsfor example as described in International Patent Specification No.WO86/01533 and European Patent Specification No. 392745.

Chemical synthesis of the hybrid proteins according to the invention maybe achieved by coupling appropriately functionalised antibody, carrierprotein and, where appropriate, bridging groups in a predeterminedorder. Standard chemical coupling techniques may be employed utilisingstarting materials containing one or more reactive functional groupssuch as thiols, acids, thioacids, anhydrides, acid halides, esters,imides, aldehydes, ketones, imines and amines. The starting antibody andcarrier protein may be readily obtained from natural sources and/or byrecombinant DNA techniques as described previously. Suitable bridginggroups, for example in the 10 Å-20 Å length range as described above,are either commercially available [see for example Pierce & Warriner(UK) Ltd., Chester, UK] or may be obtained by simple functionalisationof known readily available chemical using convention chemistry.

Thus in one general approach, a homo- or heteropolyfunctional e.g. bi-or trifunctional bridging group, may first be coupled to either theantibody or carrier protein and the resulting product coupled asnecessary to the remaining component(s) to provide the hybrid protein ofthe invention. The coupling reactions may be performed using standardconditions for reactions of this type. Thus for example the reaction maybe performed in a solvent, for example an organic solvent, anaqueous-organic solvent or, especially, an aqueous solvent at or aroundambient temperature up to around 70° C. Preferably, to avoid unwantedpolymerisation in the first coupling reaction the homo- orheteropolyfunctional bridging group is employed in excess concentrationrelative to the antibody or carrier protein. Similarly in the secondcoupling reaction, the antibody or carrier protein is preferablyemployed in excess concentration to the product of the first couplingreaction. Illustrative reactions are described in detail in the Exampleshereinafter for the preparation of proteins according to the inventionand these may be readily adapted using different starting materials toprovide other compounds of the invention.

The following Examples illustrate the invention. In these reference ismade to various figures which are:

FIG. 1.

SDS PAGE of RSA-Fab′ Conjugate.

The RSA-Fab′ conjugate was run under both reducing and non-reducingconditions. Approximate apparent molecular weights were estimated bycomparison with standard proteins run on the same gel under reducingconditions. The molar ratios were determined by N-terminal sequencing ofbands blotted from gels onto PVDF membrane.

FIG. 2.

Pharmacokinetics of RSA-Fab′ Conjugate or Controls in the Rat.

Proteins were ¹²⁵I-labelled prior to injection into 6 rats.Radioactivity present in blood sample taken at intervals was quantifiedby gammacounting. Results of analysis by Winnonlin are shown. The unitsfor the plasma half-life in α and β phases (t_(1/2)α and t1/2β,respectively) are hours (h). The areas under the plasma concentrationtime curves (AUC, 0-∞) are in the units h^(•) % dose. Data plotted asRSA-Fab′ conjugate (correction factor) have been corrected from data forRSA-Fab′ conjugate to allow for instability of labeled protein, asdecribed in the text.

FIG. 3.

Pharmacokinetics of RSA-Fab′ or Fab′-Cys in the Rat (Assayed by ELISA orCytokine Neutralisation).

Unlabeled proteins were injected into 2 rats, and subsequentlyquantified in samples of plasma by ELISA. In the case of the conjugatethe ELISA employed binding of ligand (TNF) and of anti-albumin antibody,and in the case of the Fab′-cys control it employed binding of TNF andof anti-kappa L chain, as described in the text. The units for theplasma half-life in α and β phases (t_(1/2)α and t_(1/2)β, respectively)are hours (h). Alternatively, samples were assayed by neutralisation ofTNF activityin the L929 assay. The areas under the plasma concentrationtime curves (AUC, 0-∞) are in the units h^(•) % μg/ml. Curves forRSA-Fab′ overlay each other, and curves for Fab′-cys do likewise. Errorbars=s.e.m.

FIG. 4.

Construction of pPIC(scFv-HSA)

Flow chart of construction method.

FIG. 5.

Reducing SDS PAGE of HSA-scFv Fusion Proteins.

A sample of each fusion protein before and after Blue sepharosechromatography was run under reducing conditions, together with standardmolecular weight markers and standard RSA on the same gel.

FIG. 6.

Pharmacokinetics of HSA-Fab′ Fusion Proteins or HSA in the Rat.

Proteins were ¹²⁵I-labelled prior to injection into 6 rats.Radioactivity present in blood sample taken at intervals was quantifiedby gammacounting. Results of analysis by Winnonlin are shown. The unitsfor the plasma half-life in α and β phases (t_(1/2)α and t_(1/2)β,respectively) are hours (h). The areas under the plasma concentrationtime curves (AUC, 0-∞) are in the units h^(•) % dose.

FIG. 7.

Structure of Trimaleimide Crosslinking Agent.

This reagent was used in generation of RSA-F(ab′)₂ conjugate.

FIG. 8.

Reducing SDS PAGE of HSA-F(ab′)₂ Conjugate.

Purified sample and standard proteins were run on the same gel underreducing conditions. The molar ratios were determined by N-terminalsequencing of bands blotted from gels onto PVDF membrane.

FIG. 9.

Scatchard Plots of Binding of RSA-F(ab′)₂ or F(ab′)₂ Control to CellMembranes.

2 values for K_(D) were calculated for each of the (biphasic) curves, asshown. Each phase of each curve is shown as a straight line

EXAMPLE 1

Fab′-Albumin Conjugate.

Methods

Preparation of Anti-TNF Fab′

Recombinant anti-TNF Fab′ was produced in E. coli, and prepared from theperiplasm by the methods described in International Patent SpecificationNo. WO98/25971.

Conjugation of the Anti-TNF Fab′ with Rat Serum Albumin

Rat serum albumin (RSA, fraction V, Sigma, code no. A-6272) wasdissolved to 6.7 mg/ml (0.1 mM) in sodium acetate, 0.1M, pH 5.9.Dithiothreitol solution (100 mM in the same acetate buffer) was added togive a final dithiothreitol concentration of 0.3M, so giving a 3-foldmolar excess over RSA. The mixture was incubated at 37° C. for 40 min.The mixture was then subjected to chromatography on Sephadex G25M usinga PD10 column (Pharmacia, code no. 17-0851-01, used as permanufacturer's instructions), thus removing dithiothreitol andexchanging the buffer for sodium phosphate, 0.1M, pH 6, 2 mMethylenediamine-tetraacetate (EDTA). The RSA concentration at that stagewas 70 μM.

1,6 bismaleimidohexane (BMH, Pierce, code no. 22330) was dissolved to7.736 mg/ml (28 mM) in dimethylformamide. The BMH solution was added tothe reduced RSA solution to give a 21-fold molar excess of BMH over RSA.The mixture was incubated at 21° C. for 100 min, then subjected tochromatography on G25M in a buffer of sodium phosphate, 0.1M, pH 6, 2 mMEDTA. The concentration of derivatised RSA was 46 μM at that stage. Thesolutions of derivatised RSA and the anti-TNF Fab′ (187 μM) in sodiumphosphate, 0.1M, pH 6, 2 mM EDTA were mixed to give a molar ratio,RSA:Fab′:1:1.3 (which, corrected for derivatisation of RSA and reductionof Fab′ thiol gave a ratio, derivatised RSA:reduced Fab′:1:1.4). Themixture was incubated at 21° C. for 2 h, although reaction wasessentially complete within 1 h. The mixture was then stored at 4° C.until subjected to purification procedures.

Conjugation of Fab′ with Cysteine

The method for preparation of control molecule, anti-TNF Fab′ covalentlylinked by BMH via thiols to cysteine (instead of RSA) was essentiallythe same as for preparation of conjugate (see above). 20 μM Fab′ wasreacted at 21° C. for 95 min with a 40-fold molar excess of BMH (addedas a solution in dimethylformamide). After chromatography on G25M (toremove BMH) the derivatised Fab′ was reacted with cysteine (Sigma, codeno. C4820) at a molar ratio Fab′:cysteine free thiol:1:4.5. Afterreaction at 21° C. for 160 min, the sample was stored at 4° C. prior topurification of the Fab′-cys product.

Purification of the Conjugate

The reaction mixture was first subjected to chromatography on Gammabindplus (Pharmacia), a matrix that has affinity for Fab′. A 5.3 ml columnwas equilibrated in a buffer of sodium phosphate, 0.1M, pH 6, 2 mM EDTAat a flow rate of 2 ml/min. All chromatography was at a temperature of21° C. The sample was applied to the column at a flow rate of 1 ml/min,and the column then washed in the same buffer (sodium phosphate, 0.1M,pH 6, 2 mM EDTA) until the baseline was restored. Adsorbed protein waseluted by application of a buffer of acetic acid, 0.5M, made to pH 3 byaddition of sodium hydroxide. The whole eluent was collected infractions and each fraction analysed by SDS PAGE (using both reducingand non-reducing conditions). As expected of this affinity matrix,unconjugated Fab′ eluted in the pH 3 buffer, whereas the unconjugatedRSA did not bind to the matrix at all, and emerged in the flow-throughduring sample loading. Conjugation of a single Fab′ to one RSA moleculeclearly affected its binding to the protein G on the matrix, for theconjugate emerged in the flow-through, just slightly later than (andoverlapping with) the unconjugated RSA. The fraction containing theconjugate was subjected once more to chromatography on Gammabind plus(as above), in order to separate more RSA from it. The fractionscontaining conjugate (and some traces of RSA) were concentrated in astirred cell (Amicon, 10 kDa nominal molecular weight cut-off membrane).

The contaminating RSA was removed from the preparation by gel permeationchromatography on a GF250 HPLC column of size 2×23cm (using a HewlettPackard 1090 HPLC). The column was equilibrated and eluted in a bufferof sodium phosphate, 0.2M, pH 7, at a flow rate of 3 mL/min, at 21° C.The sample of concentrated conjugate (and RSA) was chromatographed andelution monitored at 280 and 220 nm. Fractions corresponding to observedpeaks were collected and analysed by SDS PAGE. Those fractionscontaining the conjugate (which was completely resolved from thelater-eluting RSA) were pooled and concentrated in a stirred cell(Amicon, 10 kDa nominal molecular weight cut-off membrane). The solutionwas stored at 4° C., with sodium azide added to 0.05% (w/v), to act as apreservative.

Purification of the Fab′-cys

The reaction mixture containing Fab′-cys product was diluted 5-fold in abuffer of sodium acetate, 50 mM, pH 4.5, then loaded onto a Mono Scolumn (of 1 mL volume), using an FPLC (Pharmacia) apparatus. Adsorbedproteins were then eluted in a gradient of 0 to 250 mM sodium chloridein sodium acetate, 50 mM, pH 4.5. Elution (1 ml/min at 21° C.) wasmonitored by absorption at 280 nm. Collected peaks were analysed by SDSPAGE.

Radiolabelling of Proteins

Proteins were labeled at the ε-amino groups of lysyl residues, using¹²⁵I-labeled Bolton and Hunter reagent (Amersham International, code no.IM5861). Proteins were dissolved or diluted in a buffer of borate, togive a final borate concentration of 0.1 M, pH 8. A solution (of between300 and 370 μL) containing 300 μg protein was then mixed with 20 μLBolton and Hunter solution in propan-2-ol (containing 9 MBq of ¹²⁵I) Themixture was incubated at 21° C. for 15 min, then the reaction wasquenched by addition of 60 μL solution of glycine, 1M, in borate, 0.1M,pH 8.5. After approximately 5 min reaction at 21° C., the reactionmixture was chromatographed on Sephadex G25M using a PD10 column(Pharmacia, code no. 17-0851-01, used as per manufacturer'sinstructions). In doing so, the buffer was exchanged for phosphatebuffered saline. The specific activity of each preparation wascalculated from estimates of protein concentration (see AnalyticalProcedures) and of radioactivity, and were typically in the range 0.45to 0.54 μCi/μg. The radiolabeled samples were used directly afterlabeling.

Analytical Procedures

Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS PAGE)utilised precast SDS gels (Novex), 1 mm thick and of acrylamideconcentration 4 to 20%, run as per manufacturer's instructions. Gelswere stained by soaking for 1 h in Coomassie BBG in perchloric acid(Sigma, code no. B-8772), followed by washing in water. Variousmolecular weight standards were used in order to derive approximatemolecular weights (apparent) for sample proteins. These standards wereMark 12 and Seeblue unstained and prestained markers, respectively(Novex). For western blots, SDS PAGE was followed by blotting topolyvinylidene difluoride membrane (Millipore), with detection of the Fdfragment of the Fab′ by sheep anti-human IgG(Fd) IgG fraction (TheBinding Site, code no. PC075) followed by peroxidase-affinipure F(ab′)₂fragment of rabbit anti-sheep IgG, Fc fragment (Immunoresearch, code no.313036046) and visualisation by use of chemiluminescence (ECL; AmershamInternational). For autoradiography, the SDS PAGE was followed byexposure of the gel to photographic film (Hyperfilm MP; AmershamInternational). For imaging of radiolabeled samples on gels (andsubsequent quantification) the gels were exposed to high resolution orgeneral purpose screens and processed in the Canberra Packard Cyclonesystem, using Optiquant software.

Quantification of protein solutions was by absorption at wavelength 280nm in a 1 cm cell, using absorption coefficients (for 1 mg/mL solutionin a 1 cm cell) of 1.43 for Fab′ or F(ab′)₂, and of 0.58 for RSA. Acoefficient of 1.0 for RSA-Fab′ conjugate was calculated from those ofRSA and Fab′, weighted in accordance with the constituent masses of thetwo components.

The concentration of free thiol in a protein solution was measured byadding 1/9 volume of 4,4′-dithiodipyridine (5 mM, final concentrationtherefore was 0.5 mM) in phosphate buffered saline. After 10 min at 21°C., the absorbency at 324 nm was measured in a 1 cm cell. The absorbencyof a buffer-only blank sample was subtracted from this value, and thisfigure multiplied by 56.1167 to give the result in μM thiol (this beingfurther corrected for any dilution of the original sample).

N-terminal protein sequencing was performed as per manufacturer'sinstructions on a model 470A protein sequencer with on-line 120 A HPLCand 900A data analysis system (Applied Biosystems). Protein in solutionwas adsorbed to polyvinylidene difluoride membrane in a Prosorb device(Applied Biosystems). Proteins from SDS PAGE were blotted topolyvinylidene difluoride membrane (Immobilon PSQ, Millipore), andprotein bands detected by staining by 0.1% (w/v) Ponceau S (Sigma, codeno. P-3504) in 1% (v/v) acetic acid for 1 min, then destaining thebackground in water. Bands were excised and sequenced directly.

Surface plasmon resonance study of interactions with ligand wasperformed on a BIACORE 2000 (Biacore AB), as per manufacturer'srecommendations. Sensor chip surface coated by goat anti-human F(ab′)₂antibody (Jackson ImmunoResearch Lab. Inc.), which binds to the lightchain, was used to bind conjugate, Fab′ or IgG, whose binding to ligand(TNF) from solution was then measurable.

ELISA's were performed as follows below, with steps interspersed bywashing in 0.1% Tween 20 in phosphate buffered saline. Microtitre platewells were coated with cytokine antigen. Non-specific binding sites werethen blocked by incubation of the wells with a 5% (w/v) solution ofdried skimmed milk (“Marvel”, Premier Beverages, UK) in phosphatebuffered saline, for 1 h. Samples or standards were diluted in bovineserum albumin, 1% (w/v) in phosphate buffered saline, and then incubatedin the wells for 1 h at 21° C. Quantification was by absorption at 630nm, generated from 3,2′5,5′ tetramethyl benzidine (120 μM in 10 mMacetate, pH 6) as a result of the activity of peroxidase, followingeither of:

(i) For detection of albumin-Fab′ conjugate, incubation of each wellwith rabbit anti-rat albumin (Cappel, product no. 55711, diluted 1 in4000 in 1% bovine serum albumin in phosphate buffered saline), followedby goat anti-rabbit immunoglobulin Fc-peroxidase conjugate (Jackson,product no. 111-036-046, diluted 1 in 5000 in 1% (w/v) bovine serumalbumin in phosphate buffered saline).

(ii) For detection of Fab′, incubation in goat anti-human kappa lightchain (Southern Biotechnology Associates, Inc., product no. 2060-01,diluted 1 in 5000 in 1% bovine serum albumin in phosphate bufferedsaline), followed by donkey anti-goat immunoglobulin (H+L)-peroxidaseconjugate (Jackson, product no. 705-035-147, diluted 1 in 5000 in 1%(w/v) bovine serum albumin in phosphate buffered saline).

Assay format (i) gave a linear response in the range 40-500 ng/ml ofconjugate, and assay format (ii) gave a linear response in the range5-100 ng/ml of Fab′

To assay the neutralisation of TNF activity, a monolayer of mouse L929cells was grown in normal RPMI 1640 plus glutamine and 10% (v/v) foetalcalf serum. TNF added in presence of actinomycin D (1%, w/v) with orwithout sample/anti-TNF. Cell death caused by TNF was monitored by MTTassay: 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT; Sigma, code no. M-2128) was added to a final concentration of 50μg/ml in the medium of treated cells and incubated at 37° C. for 4 h.Reaction was stopped and the brown colour produced by live cells wassolubilised by a solution of SDS, 20% (w/v) in 50% dimethylformamide inwater, at pH 4.7 (by addition of 50% acetic acid, 1M, in hydrochloricacid, 1M) and then quantified by measuring absorbance at 570 nm. Havingsubtracted the absorbance at 630 nm, the degree of cell survival (and sothe concentration of active TNF present) could be assessed by comparisonwith samples of cells treated by standard amounts of TNF in the absenceof TNF-neutralising activity.

Incubation of Protein in Plasma or Blood in vitro

Rat plasma and blood (heparinised) were prepared fresh. To 100 μl wasadded 12 μl of ¹²⁵-labeled protein solution. Incubation was at 37° C. ina capped 0.5 ml tube, with 0.5 and 2 μL samples withdrawn at intervalsfar SDS PAGE and autoradiography.

Unlabeled conjugate or Fab′-cys (2 μl of 0.9 mg/ml solution) was mixedwith 100 μl fresh rat plasma and incubated at 37° C. in a capped 0.5 mltube. Samples of 2 μl were withdrawn at intervals for analysis bywestern blotting.

Pharmacokinetic Analysis of the Conjugate

Male Wistar rats (of approximately 250 g each) each received 20 μg of¹²⁵I-labeled, or 180 μg of unlabeled protein in solution in phosphatebuffered saline, being injected into a tail vein. Samples of blood weretaken from the tail artery at intervals thereafter, with plasma preparedby heparinisation and centrifugation. Samples were analysed as describedabove (see Analytical Procedures) and radioactivity in whole blood wascalculated as cpm/g blood. The percent injected dose (% ID) wascalculated for each individual rat, based on standards and expressed as% ID/ml total blood volume. Groups of 6 or 2 rats were used for study of¹²⁵I-labeled or unlabeled protein, respectively.

Data were analysed by WinNonlin software in order to determine thepharmacokinetic values quoted. A two compartment model was used for thisanalysis unless otherwise stated.

Results

Characterisation of the Conjugate

It is known that although serum albumin has one cysteinyl residue thatis not engaged in a disulphide bond (residue 34 in the mature humanalbumin sequence), many of the molecules in a preparation of albumin donot possess a free cysteinyl thiol due to formation of mixed disulphideswith molecules such as glutathione or free cysteine [Peters, ibid 1985].In accordance with this, analysis of the rat albumin solution prior toreduction showed that there was 0.15 mole of thiol per mole of albumin,i.e. only 15% of albumin molecules possessed a free thiol. Afterreduction, this was increased to 0.95 mole of thiol per mole of albumin,consistent with presence of a free thiol group in 95% of albuminmolecules, most likely at position 34 in the mature albumin sequence,since reduction under conditions such as those used here does notdisrupt any of the disulphide present elsewhere in the molecule [Peters,ibid 1985]. Subsequent reaction at this cysteinyl residue allowedattachment of another molecule at a specific site, and in a 1:1 molarratio, without concomitant production of other products of higher ratioof albumin:other molecule that would need to be removed subsequently.Co-ordinates for the crystal structure of human serum albumin have beendeposited in the Brookhaven database (as entry 1AO6, by S. Sugio, S.Mochizuki and A. Kashima). Inspection of this model shows that the freecysteinyl residue at position 34 lies in a groove between two helices,but it is not clear from this how long a spacer arm on a crosslinkingagent should be in order to work effectively. Absence of albuminhomo-dimer would be advantageous in a production process since the yieldof hetero-dimer then would be greater, and also there would be no needfor an additional step to remove the homo-dimer. It was found that thecross-linking agent used in the present example produced thisadvantageous result. Reaction of the reduced albumin with a 20-foldmolar excess of BMH caused derivatisation of 80% of these albuminmolecules. Albumin homo-dimers were not formed during albuminderivatisation (or subsequent reaction with other protein molecules).The cross-linking agent spacer arm, of length 16.1 Å, was long enough toreach from the cysteinyl thiol at position 34 to the surface of thealbumin molecule, where it was able to react with the free thiol on aFab′ molecule. It was not long enough to penetrate the equivalent grooveon a second albumin to reach the free thiol there, and so production ofhomo-dimers was avoided.

The derivatised albumin and Fab′ were mixed in the molar ratio1:1.4:albumin:Fab′. SDS PAGE analysis of the products at the end ofreaction indicated a yield of albumin-Fab′ conjugate of the order of 20to 30%, though this would be expected to be improved by optimisation ofreaction conditions. The conjugate was purified from the reactionmixture by affinity chromatography, the conjugate surprisingly notbinding to the matrix as did Fab′, but eluting marginally later than didalbumin, which did not bind at all. Having removed unreacted Fab′,albumin was separated from the conjugate by size exclusion. The finalpreparation of conjugate was analysed by SDS PAGE (FIG. 1), whichindicated (by apparent molecular weights) the linkage of one Fab′ peralbumin molecule. On non-reduced SDS PAGE the main band was a conjugateof 1 RSA with 1 Fab′ molecule, as determined by N-terminal sequencing ofthe protein in the band. Approximately 37% of the conjugate was presentas a higher molecular weight band that also had a RSA:Fab′ ratio of 1:1.This was assumed to be dimer material, analogous to the observedoccurrence of albumin polymers in untreated preparations of albumin.Lesser amounts (approximately 2% of total material) of conjugate missingL chain, and a putative small derivative of the conjugate (approximately4% of total) were also present. N-terminal protein sequence analysis ofthe main band on reducing SDS PAGE showed it to be RSA-Fd (i.e. Fab′ Hchain), so that the linkage was specifically via the Fab′ Fd and not viathe L chain. Thus the 1:1 stoichiometry of conjugation, was confirmed.

Conjugation of Fab′ to cysteine (to produce the control molecule,Fab′-cys) gave good yield—virtually all the Fab′ derivative reacted withcysteine with only traces of F(ab′)₂-like material of greater thanapproximately 55 kDa (apparent) on non-reducing SDS PAGE. This trace ofmaterial eluted just after the Fab′-cys product, which eluted at asodium chloride concentration of about 75 mM on Mono S chromatography,and was excluded from it.

The ability to assay the conjugate in an ELISA that depended on theconjugate binding to TNF indicated that the conjugate retained theability to bind TNF. This was confirmed by analysis of the conjugatebinding to TNF by surface plasmon resonance using the BIACORE 2000(association and dissociation rates, and equilibrium constant—see Table1). This showed that the anti-TNF Fab′ was unaffected by conjugation toalbumin. This was also confirmed by its equal capacity forneutralisation of TNF: in an assay of L929 cell death caused by TNF theconcentration of protein to inhibit 90% of TNF activity (the IC90) forthe albumin-anti-TNF Fab′ conjugate was 14.7 pM, as compared to 18.0 pMfor anti-TNF Fab′-cysteine conjugate, 10.9 pM for anti-TNF F(ab′)₂, and11.3 pM for anti-TNF IgG4. The maintenance of Fab′ binding activity wasattributed to the intended orientation of the Fab′ binding domain awayfrom the point of conjugation to the albumin molecule, achieved bytargeting of the Fab′ hinge region as the point of attachment to theother moiety.

The control, Fab′-cys, also retained its ability to bind to, and toneutralise, TNF, as seen by its detection by ELISA and its activity inthe L929 assay.

Pharmacokinetic Analysis of the Conjugate in Rat Plasma

¹²⁵I-labeled RSA, Fab′, F(ab′)₂ and RSA-Fab′ conjugate were monitored inplasma sampled at various times over 144 h (FIG. 2). The observedβ-phase half-life of albumin was in agreement with the literature valueof about 2 days [Peters, ibid 1985]. The β-phase half-life of theF(ab′)₂, and the Fab′, were similar to that of F(ab′)₂ describedpreviously [e.g. Kitamura et al, ibid; Chapman et al, ibid], and waspreceded by rapid elimination in the α-phase, typical of such molecules.However, when the Fab′ was conjugated to RSA it persisted in circulationin the rat to a degree comparable to rat albumin. Due to a reduction ofelimination during both α- and β-phases, the conjugate showed a 35-foldgreater area under the plasma concentration curve (AUC, 0-∞) than didthe Fab′-cys control, similar to that of albumin alone. In order toensure that radioactivity detected in the plasma samples reflectedremaining conjugate, samples from one rat given labeled conjugate wererun on SDS PAGE and scanned by phosphorimager (data not shown). Thisshowed the persistence in vivo of intact labeled conjugate for at least120 h, and the β-phase half-life of intact conjugate was calculated tobe 32.07 h, in good agreement with the equivalent result from total ¹²⁵Idetection.

The stability of the conjugate in plasma was inspected. Incubation ofthe unlabelled conjugate in rat plasma (in vitro) at 37° C. for 68 h inthe presence or absence of a variety of protease inhibitors, monitoredby SDS PAGE and western blotting, indicated that the conjugate wasstable in rat plasma. Conjugate that had been labeled by ¹²⁵I wasincubated in vitro in phosphate buffered saline (pH 7) for up to 10 daysand was also found to be stable.

However, incubation of the 125I-labeled conjugate at 37° C. in ratplasma or blood in vitro indicated that the molecule was not completelystable, some molecules suffering cleavage at or near the point oflinkage between the albumin and the Fab′ molecule. Since unlabeledconjugate was stable, the observed instability was attributed tomodification of the protein by the presence of ¹²⁵I or by the labelingprocess. Despite an apparent instability in these conditions, intactmaterial remained even after 168 h incubation in vitro. The integrity ofthe conjugate in vivo was assessed by SDS PAGE of plasma samples,followed by quantification of the intact conjugate by phosphorimagerscanning, and as in experiments in vitro, a small instability of¹²⁵I-labelled conjugate was noted. The observed results from in vivoexperiments could be adjusted accordingly to reflect the quantity ofintact, labeled conjugate only. The adjusted data gave the result shownin FIG. 2 (see conjugate (correction)), which was that even the slightlyunstable labeled version of the conjugate had an AUC that was 17-foldgreater than the Fab′-cys control.

The conclusion that data derived from the use of ¹²⁵I-labeled conjugaterepresent an underestimate of the longevity of the unlabelled conjugatein vivo was supported by an experiment in which the conjugate was notlabelled. Unlabeled protein was monitored by two forms of ELISA and bybiological activity (TNF neutralisation in the L929 assay). Results fromuse of the three assays in samples from two rats were similar to eachother (FIG. 3), showing that RSA-Fab′ was stable in vivo for protractedperiods. However, Fab′-cys was eliminated rapidly, such that too fewdata were obtained by ELISA to allow use of a two compartment model, andthe AUC estimate given in FIG. 3 is by use of a one compartment model.Too few data were obtained by use of activity assay to allow anymodeling at all. The RSA-Fab′conjugate's AUC was about 200-fold greaterthan that of the Fab′-cys control.

It has been shown that it is possible to prepare an IgG antibody Fab′fragment, chemically crosslink it to albumin in vitro with retention offull binding ability (having the same affinity as seen in the wholeantibody), and demonstrate that it has a significantly longer half-lifein vivo than does the unconjugated Fab′. The conjugate contained oneFab′ per albumin molecule. The β-phase half-life of the conjugate in therat was about twice that of the unconjugated Fab, being closer to thatof unconjugated albumin, and the area under the curve was about 200-foldgreater for the conjugate than for the unconjugated Fab′. Thus, theconsequence of conjugation of Fab′ to albumin was a significantprolongation of its biological activity in vivo, allowing (in thepresent case) prolonged anti-cytokine therapy.

EXAMPLE 2 Genetic Fusion of scFv to Albumin

Methods

Construction of Plasmids Coding for scFv-Human Serum Albumin FusionProteins.

Plasmid pPIC(scFv) contained the gene for an anti-TNF scFv, comprisingthe variable domains in the order V_(L)-V_(H). This plasmid was cleavedwith restriction enzymes KpnI and NotI to generate a ‘vector’ DNAfragment which was purified following agarose electrophoresis. Into thiswas ligated a PvuII-NotI fragment of DNA containing the mature HSA gene,together with a 200 bp linker encoding a direct in-frame fusion betweenthe C-terminus of the scFv and the N-terminus of HSA. This procedure issummarised in FIG. 4. The linker or junction fragment also introduced aunique ClaI restriction site to facilitate the construction of a furtherseries of genes encoding fusion proteins containing different spacers tophysically separate the scFv and HSA motifs. Annealed oligonucleotidecassettes were used to introduce the following spacers between the KpnIand ClaI sites:

-   -   1. A sequence encoding 3 repeats of the Gly₄Ser sequence.    -   2. A sequence derived from the flexible N-terminal end of the        human IgG1 hinge region.

In addition, a gene encoding a further fusion protein was generated inwhich the C-domain from human Ig-kappa light chain was used as a spacerto separate the scFv (this time in the V_(H)-V_(L) orientation) from theHSA. The construct nomenclature was: scFv-HSA; scFv-G4S-HSA;scFv-UHL-HSA; scFv-C_(K)-HSA, respectively.

Expression of Fusion Proteins in Yeast

These plasmids were linearised and transfected into competent Pichiapastoris cells. This was done using an “EasySelect Pichia Expression”kit (Invitrogen) by following the manufacturer's instructions.Transformants were selected by resistance to Zeomycin (500 g/ml)incorporated in the YPDS agar used to grow them. Transformants wereconfirmed as being Mut⁺, that is, capable of utilising methanol as thesole carbon source, though sensitive to the presence of excess methanol.Two colonies of each of the 4 types of transformant were picked andgrown in shake flask culture in BMGY medium, as per “EasySelect PichiaExpression” kit instructions. Each culture was 25ml in a 25 ml flask,incubated at 30° C., shaking at 225 rpm. After 16-18 h, when thecultures had grown to give an optical density at 600 nm (OD600) ofbetween 2 and 6, cells were harvested by centrifugation and resuspendedin 30-40ml of BMMY medium (which contains methanol as carbon source), togive an OD600 of 1. Incubation was then continued with shaking andoccasional addition of methanol in order to maintain a methanolconcentration of approximately 0.5% (v/v). After 84 h incubation thecultures were centrifuged and the supernatants retained for use inpreparation of expressed and secreted fusion proteins.

Purification of Fusion Proteins.

Expressed proteins were purified from the yeast culture medium afterclarification by centrifugation. The medium was subjected tochromatography on a matrix of Blue sepharose (Pharmacia), which acted asan affinity matrix for albumin-containing proteins, as follows below.Each clarified supernatant was mixed with one volume of phosphatebuffered saline, pH 7, and the pH of the mixture increased from about6.5 to about 7 by addition of a small volume of sodium hydroxidesolution (2M). The mixture was then applied to a 1.5 ml column of Bluesepharose, pre-equilibrated in phosphate buffered saline, pH 7. The flowrate at all stages was 0.5 ml/min, and the temperature was 21° C.Following application, the column was washed by phosphate bufferedsaline until a stable baseline was achieved, and then bound protein waseluted by the eluent sodium thiocyanate, 0.2M in phosphate bufferedsaline. Elution was monitored by absorbtion at 280 nm wavelength. Elutedprotein was collected, concentrated, and its buffer exchanged tophosphate buffered saline by use of a stirred cell concentrator (Amicon,using a 10 kDa nominal molecular weight cutoff filter membrane). Proteinwas quantified by absorbance at a wavelength of 280 nm and use oftheoretical absorbtion coefficient that was calculated for each fusionprotein by use of the program ProtParam (found at the ExPasy website).

Radiolabeling of Proteins

Fusion proteins with no linker, with G4S linker or with upper hingelinker, and unmodified human serum albumin were labelled by ¹²⁵I asdescribed in Example 1, above.

Analytical Procedures

Proteins were characterised and their pharmacokinetics in rats analysedas described in Example 1.

Results

Characterisation of Fusion Proteins

All four scFv-human serum albumin (HSA) fusion proteins were expressedfrom Pichia pastoris, being secreted into the medium at the followingrates, as determined by absorbance at 280 nm of protein solutionsprepared by chromatography (mean of 2 experiments): scFv-HSA (nolinker), 9.0 μg/ml; scFv-G4S-HSA, 8.5 μg/ml; scFv-UHL-HSA, 7.5 μg/ml;scFv-CK linker, 7.8 μg/ml. Bands corresponding to the expressed proteinscould be seen upon SDS PAGE of unfractionated yeast culture medium (e.g.FIG. 5). No development was undertaken with the aim of improvingexpression levels. These data suggested that the order of the variabledomains in the scFv portion did not significantly affect the levels ofexpression of the fusion protein.

Reducing SDS PAGE (FIG. 5) of prepared fusion proteins after only Bluesepharose chromatography showed each to contain one principle band andone minor, where the major species accounted for 91% of all protein inthe scFv-HSA preparation, 75% in the scFv-G4S-HSA preparation, 80% inthe scFv-UHL-HSA, and 83% in the scFv-CK-HSA preparation, as estimatedby scanning of brilliant blue G-stained gels. The principle band in eachpreparation had an apparent molecular weight equal to the theoreticalmolecular weight calculated from the translated DNA sequence. The minorband in each migrated with standard human serum albumin on the same gel,indicating minor cleavage event(s) at the sequence between albumin andscFv or scFv-hinge/spacer sequence. N-terminal sequencing of this minorproduct in the scFv-G4S-HSA preparation showed that it did indeed havethe N-terminus of mature HSA. N-terminal sequence analysis of the fusionproteins showed them to have the expected scFv N-terminus, in each casepreceded by the sequence EAEA, which had been included in the fusionprotein in order to alleviate possible problem(s) of steric hindrancerendering the cleavage of the pro-sequence inefficient (as discussed bySreekrishna, K. et al (1997) Gene, 190, 55-62). This EAEA spacersequence was not subsequently removed by yeast diaminopeptidaseactivity, however. Nevertheless, this N-terminal EAEA extension did notobviously inhibit expression and secretion of the heterologous proteins.It is likely that the EAEA sequence could be deleted from the fusionprotein in order to generate a protein of authentic scFv (or other)N-terminus.

Blue sepharose chromatography was shown to be efficient in purificationof albumin-containing proteins in a single step. These proteins includedminor products of proteolysis in the present cases, but no optimisationwas undertaken here. Thus, proteolysis would be expected to be reducedby such means as optimisation of culture conditions, inclusion ofprotease inhibitors, and/or expression in protease-deficient strains ofyeast (for instance, as described by Gleeson, M. A. G. et al in “PichiaProtocols” [eds. Higgins, D. R. and Cregg, J. M.), pub. Humana Press,Totowa, N.J. (1998), pp 81-94]. In terms of industrial processes, thecost of Blue sepharose is very low, certainly considerably lower thanany other commercially-available affinity matrix. Thus, the use ofalbumin as part of a fusion protein is doubly beneficial, since it cannot only add to the protein such properties as long serum half-life (seeExample 1 or below), but also allow rapid, low cost purification. Theability of the fusion proteins to bind ligand was confirmed by Biacoreanalysis. This showed that the association and dissociation rates andK_(D) of the 4 types of fusion protein were similar to each other and toother forms of the anti-TNF antibody, including whole IgG (Table 1).

Pharmacokinetic Analysis of the Fusion Proteins in Rat Plasma

The fusion proteins scFv-HSA, scFv-G4S-HSA and scFv-UHL-HSA were¹²⁵I-labelled, as was HSA alone. The minor component of albumin that waspresent in each of the fusion protein preparations became labeled, too.

Analyses of the distribution of label in the in the fusion protein andalbumin components of the preparations by the two methods ofautoradiography and by phosporimaging gave similar results. The mean ofthe two methods was: for scFv-HSA, 76% of label was in the fusionprotein; for scFv-G4S-HSA, 80% of label was in the fusion protein; forscFv-UHL-HSA, 40% of label was in the fusion protein. Remaining labelwas taken up by the albumin component of each preparation.

These proteins were injected into rats (n=6). Plasma samples were takenat intervals for analysis by gamma counting and by SDS PAGE followed byphosphorimaging. The distribution of label in the different samples weredetermined during the first 48 h by phosphorimaging of samples from onerat per group (replicate number, n=2 or 4). In each case there was foundto be no clear change in the distribution of label during the 48 hperiod (data not shown). Therefore, the fusion proteins were not proneto significant degradation in vivo. It was assumed that this stabilitywas maintained for the full duration of the experiment, which was 144 h,and the pharmacokinetic analysis of the proteins was based on theresults of gammacounting. The pharmacokinetics were similar for allproteins (see FIG. 6), thereby showing that the fusion proteins behavedsimilarly to unmodified human albumin in plasma in vivo. Note that inrats HSA is only a half (or less) as persistent as the homologous, rat,albumin, and a fusion of scFv with HSA would not necessarily be expectedto endow a half-life any longer than that of HSA itself. Nevertheless,the AUC for the ¹²⁵I-fusion proteins are approximately 13-fold greaterthan those for ¹²⁵I-Fab′-cys control (shown in FIG. 2).

This example shows that serum albumin may be fused to another protein(s)to generate a fusion that possesses function(s) of the other protein(s)together with the long half life of albumin. Furthermore, since fusiondid not involve albumin cysteinyl thiols, the possibility remains ofusing a fusion protein as an acceptor of one or more Fab′ molecules,linked by chemical means, as exemplified by Examples 1 and 3.Polyspecific as well as polyvalent molecules could be generated in theseways.

EXAMPLE 3

Fab′ Dimer-Albumin Conjugate.

Methods.

Preparation of the Dimer of Anti-Cell Surface Marker Fab′.

The anti-cell surface marker was engineered as a Fab′. It was expressedin E. coli and was extracted from the periplasm, as described inInternational Patent Specification No. WO98/25971.

The Fab′ interchain disulphide bond was reduced as follows: 12 ml ofFab′ at 20.93 mg/ml in 0.1M sodium phosphate, pH 6.0, 2 mM EDTA wasmixed with 240 μL 250 mM 2-mercaptoethylamine in the same buffer, andincubated for 50 min at 38° C. Diafiltration of the sample against thesame buffer removed the reducing agent. The yield of Fab′ at this stagewas 85.1%. Thiol assay showed there to be an average of 0.83 free thiolsper Fab′ molecule, and analysis by GF250 size exclusion chromatographyindicated the protein to comprise 93.1 Fab′ monomer and 6.9%disulphide-bonded F(ab′)₂.

The crosslinking agent was a trimaleimide compound, illustrated in FIG.7. This molecule also contained a metal-chelating function. A 1 mg/mlsolution of this compound was made in 0.1M sodium phosphate, pH 6.0, 2mM EDTA, and 5 aliquots of 434.8 μl each added with mixing at 5 minintervals to the above reduced Fab′ preparation. This provided a molarratio crosslinker:Fab′:1:2.56. The mixture was incubated at 21° C. for 3h, at which time 25 μl glacial acetic acid was added to lower the pH to4.5. 47ml water was added to lower conductivity and the mixture resolvedby ion exchange chromatography. The proteins were applied to a 2.6 cmi.d.×15.5 cm column (Pharmacia) containing methyl sulphonate cationexchanger (SP-Sepharose HP) at a flow rate of 10.5 ml/min, and eluted bya gradient of 0 to 250 mM sodium chloride in 50 mM acetate, pH4.5.Elution was monitored by absorbance at 280 nm. The various reactionproducts (monomer, dimer, and trimer) were identified by SDS PAGE and byGF250 size exclusion chromatography. Only that fraction containing dimerFab′ was used for the subsequent conjugation to albumin.

Linkage of Fab′ Dimer to Rat Serum Albumin.

33.71 mg of RSA was dissolved in 2.5 ml of 0.1 M sodium phosphate, pH6.0, 2 mM EDTA and mixed with 25 μL of 200 mM 2-mercaptoethylamine inthe same buffer, giving a final reducing agent concentration of 2 mM.The mixture was incubated at 37° C. for 1 h. Reducing agent was thenremoved by size exclusion chromatography on Sephadex G25M, using a PD10column (Pharmacia, code no. 17-0851-01, used as per manufacturer'sinstructions. The buffer was then sodium phosphate, 0.1M, pH 6, 2 mM(EDTA). The concentration of albumin was 143 μM.

The reduced albumin was incubated with conjugated Fab′ dimer in a molarratio of 1:1, and incubated at 21° C. for 24 h. The reaction mixture wasresolved by chromatography on Gammabind plus, as described in example 1(above), using a column of 3 ml volume. Unreacted albumin failed to bindto this matrix. Bound material was eluted by a buffer of 0.5M aceticacid, pH 3. Eluted fractions were immediately neutralised by addition oftrizma, 2M, pH 8.8. This material was further purified by Blue sepharosechromatography, as described in Example 2. Unreacted Fab′ dimer failedto bind to the matrix, and bound material was eluted by sodiumthiocyanate, 0.2M in phosphate buffered saline. Eluted fractions wereconcentrated and the buffer exchanged for phosphate buffered saline in astirred cell (Amicon) with a 10 kDa nominal molecular weight cutofffilter. Each type of chromatography was repeated in order to completelypurify the albumin-Fab′ dimer conjugate.

Analytical Procedures.

Procedures were as described in Examples 1 and 2, except that forN-terminal protein sequence determination, the automated sequencer usedwas a PE Biosystems Procise 492. Additionally, the ligand bindingcharacteristics of the conjugate were determined by Scatchard analysisof its binding to whole cells, as follows below.

The conjugate and a control protein (a dimer of Fab′ crosslinked throughthiols by bis-maleimido hexane) were labelled by attachment offluorescein. This was done by incubation of one mg protein in 1 ml of0.1M NaHCO₃ with 100 μg fluorescein isothiocyanate (Sigma F7250) addedas 10 μl solution in dimethyl sulphoxide. Reaction was for 2 h at 21° C.The reaction was stopped by addition of a molar excess of lysine.Labelled protein was separated from free fluorescein by size exclusionchromatography. The fluorescein-labelled antibody was collected inphosphate-buffered saline, and the protein component quantified byabsorption at 280 nm, while the extent of substitution was estimatedfrom absorption at 495 nm. The μM extinction coefficient used for DFMwas 0.14, for RSA-(Fab′)₂ was 0.2 and for fluorescein was 0.077. Thefluorescein-labelled control protein had a fluorescein:protein molarratio of 1.59, and the fluorescein-labelled RSA-(Fab′)₂ had 3.08fluorescein molecules per protein molecule.

The fluorescent protein conjugates were then serially diluted (1:1.4)from a top concentration of 2 μg/ml in phosphate buffered salinecontaining. 5% (v/v) foetal calf serum. The final volume per tube was250 μl. Forty thousand target cells were added in 100 μl per tube togive a final volume of 350 μl and a final highest antibody concentrationof 1.43 μg/ml. Cells were incubated with antibody at 4° C. for 3 h.Fluorescence activated cell sorting (FACS) analysis was then performedon the cells. Fluorescence signal was converted to molecules ofequivalent soluble fluorescein from a standard curve of fluorescentbeads according to the method of Krause et al., (1990) Determination ofAffinities of Murine and Chimeric Anti-α/β-T Cell Receptor Antibodies byFlow Cytometry. Behring Inst. Mitt. 87,56-67). The number of moleculesof fluorescein bound per cell was converted to the number of moleculesof antibody bound per cell by the fluorescein:protein ratio. Subtractionfrom the total number of antibody molecules per tube gave the number ofmolecules free per tube. Scatchard analysis was performed on these data.

Results.

In the non-optimised conditions used, the yields of products from thechemical crosslinking reaction were: Fab′ trimer, 30.6%; Fab′ dimer,30.7%; Fab′ monomer, 38.7%.

This dimer was used in subsequent reactions with albumin. Being producedIs by crosslinking with a tri-functional agent, the dimer possessed onefunctional group remaining on the linking moiety. This was a maleimidethat was able to react with a free thiol. A free thiol was generated inrat albumin by reduction, by a method that was different from that usedin Example 1. Assay of thiols in the albumin preparation reduced in thisway showed that there was then an average of 0.97 free thiols permolecule. Thus, a single thiol may be generated in albumin by more thanone method.

Incubation of the prepared Fab′ dimer with the reduced RSA gave a yieldof 1 mg of albumin-Fab′ dimer conjugate, as estimated by absorbance at280 nm, using a theoretical absorption coefficient calculated by theprogram ProtParam (ExPasy), after complete purification.

SDS PAGE (reduced) of the product showed the presence of two bands, ofapparent molecular weight of approximately 150 kDa and approximately 31kDa (FIG. 8). N-terminal sequencing showed these to be albumin-Fab′heavy chain dimer, and Fab′ light chain, respectively. Non-reduced SDSPAGE showed one main band of apparent molecular weight about 200 kDa.N-terminal protein sequencing showed this band to be albumin-Fab′ (i.e.heavy plus light chains) dimer. That the product of combination of RSAand Fab′ dimer consisted of one albumin molecule and two Fab′ moleculeswas confirmed by N-terminal sequencing of unfractionated final product,also. This also showed that no Fab′ light chain had dissociated duringproduction and that the covalent linkage between Fab′ and albumin wasvia the Fab′ heavy chain, only.

Ligand binding activity was retained by the conjugate, as shown by FACSanalysis of its binding to cells bearing ligand on their surface.Scatchard analysis of binding gave a 2-phase curve, with a K_(D) of 8 nMfor binding of one of the two Fab's, and 3 nM for binding of both Fab'sin the construct (FIG. 9). These values were similar to those of acontrol (Fab′ dimerised by crosslinking by bis-maleimido hexane i.e.lacking the albumin moiety), being 12 nM and 3 nM, respectively. As inthe other two Examples, maintenance of the Fab's full binding activitydespite the attachment of a large molecular weight moiety was probablydue to the targeting of the Fab′ hinge as the point of attachment, beingat the opposite end of the molecule from the antigen binding function.In the present Example, the albumin interfered with neither Fab′ towhich it was attached.

Thus, this process produced a very specific, bivalent product. Incontrast to Example 1, this was by modification of the immunoglobulinmoiety, rather than the albumin, prior to the final stage ofconjugation. Again in contrast to the other examples, a differentimmunoglobulin was used, exemplifying the general utility of theapproach. Using such approaches, linkage of albumin to otherpolyspecific immunoglobulins would clearly generate polyspecificmolecules with extended half-life in vivo.

The present results also exemplify inclusion of extra function in theconjugate. The present example is inclusion of a metal-chelatingfunction, such as might be useful for assay, diagnosis or therapy. Thechelating function in this example is on the linking moiety, and is amacrocycle that has previously been found to strongly bind metals,notably Indium, but possibly alternatively Copper, Gadolinium, Iron III,Cobalt III, Chromium III, Nickel or Aluminium.

Table 1.

Surface Plasmon Resonance Analysis of Binding of Ligand to Conjugate,Fusions and Other Immunoglobulins.

All immunoglobulins and derivatives were derived from the same anti-TNFantibody, and TNF was used as the ligand. See FIG. 10 for analysis ofbinding of RSA-F(ab′)₂. TABLE 1 Surface plasmon resonance analysis ofbinding of ligand to conjugate, fusions and other immunoglobulins k_(a),10⁵ M⁻¹s⁻¹ k_(d), 10⁻⁴ s⁻¹ K_(D), 10⁻¹⁰ M IgG 3.63 1.41 3.88 Fab' 2.790.56 2.01 RSA-Fab' conjugate 3.88 1.65 4.25 scFv-HSA 7.67 1.11 1.45scFv-G4S-HSA 6.58 1.32 2.29 scFv-UHL-HSA 6.64 1.52 2.29 scFv-C_(K)-HSA7.09 0.95 1.34

1-13. (canceled)
 14. A hybrid protein comprising an antigen-bindingantibody fragment covalently linked to an albumin molecule, wherein theantibody fragment and albumin molecule are directly linked through theC-terminal amino acid of the antibody fragment to the N-terminal aminoacid of the albumin molecule optionally through a spacing group.
 15. Ahybrid protein according to claim 1 wherein the spacing group is between1 and 100 amino acids in length.
 16. A hybrid protein according to claim1 wherein the antibody fragment is a monovalent Fab or Fab′.
 17. Ahybrid protein according to claim 1 wherein the antibody fragment is ascFv.
 18. A hybrid protein according to claim 1 further covalentlylinked to one or more effector or reporter groups.
 19. A pharmaceuticalcomposition comprising a hybrid protein, wherein the hybrid proteincomprises an antigen-binding antibody fragment covalently linked to analbumin molecule, wherein the antibody fragment and albumin molecule aredirectly linked through the C-terminal amino acid of the antibodyfragment to the N-terminal amino acid of the albumin molecule optionallythrough a spacing group, and an agent selected from the group consistingof a pharmaceutically acceptable excipient, a pharmaceuticallyacceptable diluent, and a carrier.